Solubilization of insoluble collagen fibers and reconstitution thereof



May 15, 1962 SOLUBILIZATION OF INSOLUBLE COLLAGEN FIBERS ANDRECONSTITUTION THEREOF Filed April 1, 1960 e.g. Sodium Doo'ecyl SulfatePeconstiiuted Collagen Fiber which is insoluble in hot Water Dry witheg. Acetone Dry Reconsfituted Fiber TOMIO NISHIHARA Collagen SourcelAnimal Skin Cut into 10" suare I Wash with SaliSolution'eg. (25AhClIWash with are Mixture of Collagen Fiber and Enzyme in the Buffer havingthe optimum PH value for the Enzym I Stand for 90 hrs at the temperaiurbellow 60C .9. 256

Dehalri/ Hair is or) [ls/1 with Salt Solution e.g OSNaClI Wash with Water Collagen Fiber treated with Enzyme in Dilute Acid oi PH 4.5-2

Solubiiizing 24 hrs at the tempera- Ture bellow 37C e9. 25C

2 Sheets-Sheet 1 H droiytie Enzyme e.g, Trypsin, Panerea tin CollagenSolution molecular/y disposed Separation of Reeonstitu ted Fiber fromWater by Centrifuge or Filter press Base To give ri5-9eg Na0H.NH40HOrganic Base Wash with Water and separation of Fiber from Water, re eat,3 times Wet heconstituted Fiber warm at 60C Dry with M. Ace tonGelcziim Solution I Dry Reeonstituted Fiber which is easily soluble inhot Water TOMIO N'ISHIHARA IN VEN TOR.

ATTORNEY Filed April 1, 1960 y 15,1962 TOMlO NISHIHARA 3,034,852

SOLUBILIZATION OF INSOLUBLE COLLAGEN FIBERS AND RECONSTITUTION THEREOF 2Sheets-Sheet 2 Collagen Source 6-9. Animal Skin car into io suaie Washwith SaltSoLutiO/l 9.9. 0.5 NaClI Wash with Wafer] Enzyme e.g. Pepsi/1Mixture of Collagen Fiber and Enzyme whose oplimum pH ranging from 4.5To 1 in Dilule Acid Solufion Solubilizing 48hrs at xhe lemperaturebellow 37C 6.5. 25C

Collagen Solution mo/eeula rly disposed Anionic Centrifuge Base 7?) giyeHweg De lergent NaOH, NH40H e.5.5od/um Separation of Reconsliluzed FiberOman/c Base Dodecyl from Wafer by Cenlrifuge 0r i/ler press Sulfale Washw/lh Wafer and separa f/an keconslilufed Collagen H'ber of Fiber fromWafer, repeat3 limes l1 s s 1 hi lr W [6/1 I m 0 U 6 mhotwae WetReconsfltutedfiber Dry wirh e9. A ela/7a Dry Recanslituzed Fiber Wafm at60 C y I 89 Ce 0 Ge/aiing S l tion Dry Reconsfizuted Fiber which iseasily soluble in hot Water 2 Z TOMIO NISHIHARA INVENTOR.-

ATTORNEY ilnited rates senses Patented May 15, 1962 ice 3,034,852SULUBILHZATION F ENSOLUBLE C(ELLAGEN FIBERS AND RECQNSTITUTION THEREQFToniio Nishihara, Tokyo, Japan, assignor to The Japan Leather Mfg. Co.,Ltd, Tokyo, Japan, a corporation of Japan Filed Apr. 1, 1960, Ser. No.19,172 Claims priority, application Japan Jan. 26, 1960 9 Claims. ((11.its-54) The present invention relates to the solubilization of collagenfibers previously considered insoluble. More particularly, the presentinvention relates to solubilization of collagen fibers, a fibrousprotein which constitutes the principal element of connective tissue inanimals. Previously, it was considered impossible to form a solution ofsuch collagen without either converting it to a gelatin or else alteringits inherent molecular structure from a helical, rigid rod type moleculeinto a foldable random coil structure which was accomplished either byheating at a temperature above 50 C. or by the use of a chemicaldenaturing agent, e.g. potassium thiocyanide, calcium chloride, urea,etc. But since 1927 it has been known that a small percentage of a givenquantity of collagen can be dissolved in a solution of dilute acid oralkali or neutral salt Without altering the inherent structure of thecollagen, that is, the helical structure of the rigid rod type, and thatthe original fiber can be reconstituted from said solution by anysuitable method. Collagen in this state has been named soluble collagen.However, such soluble collagen makes up only a small percentage of thetotal amount, the exact percentage varying sligt-hly depending on theage, the portion of body, or the kind of animal, and the greater part ofthe collagen has up to now been considered as insoluble.

One object of this invention is to provide a method for obtaining a goodyield, in soluble form, of that collagen previously considered insolublewithout denaturing the collagen (i.e. without destroying the helical,rigid rod molecular structure) and in such form that the original fibercan be reconstituted in good yield. Thus, the object of this inventionis to prepare a soluble collagen on an industrial scale. Collagen films,collagen fibers, collagen fabrics and collagen sponges can be obtainedfrom the collagen solution thus obtained. Furthermore, if the collagenfiber which has been solubilized and then reconstituted is suspended inwater and heated at a temperature of 5 to 70 C., so as to dissolve ittherein, one obtains in 100% yield a homogeneous gelatin having higherpurity and higher freezing and melting points than prior art collagengelatins. Moreover, this gelatin forms far more quickly than do those ofthe prior art.

I have discovered that so-called insoluble collagen can be dissolvedwithout denaturation to form a solution of uniform molecular Weight, byreacting the insoluble collagen With a 'hydrolytic enzyme at atemperature below 60 C., its shrinkage temperature, and then extractingthe collagen with a dilute acid solution at a temperature below 37 (3.,its denaturation temperature. Moreover, the original fiber can bereconstituted in 100% yield from the collagen solution so obtained,using any known method, e.g. neutralization, dialysis, ion exchange,addition of a surface-active agent, or extraction with an organicsolvent such as acetone or alcohol. The hydrolytic enzymes utilized inaccordance with the present invention are the proteolytic enzymes, thatis, pancreatin, trypsin and pepsin and their specific employment isshown in the following examples.

This collagen solution of uniform molecular weight in which the proteinretains its inherent molecular structure can be obtained only byextracting the hydrolytic en zyme-treated collagen with a dilute acid ata temperature below 37C. Some of the prior art methods of reconstitutingthe fiber, e.-g., dialysis of a water or disodiurn hydrogen phosphatesolution, or addition of a sodium salt such as the chloride, citrate,acetate, etc., are inconvenient in that they require an extended timeboth for reconstituting the fiber and for washing the reconstitutedfiber. By contrast, if a surface active agent is used, theseinconveniences are avoided and the fibers are simultaneouslyreconstituted and purified.

In the accompanying drawings FIGURES 1 and 2 are representatiom in flowsheet form of embodiments of the invention hereinafter described.

The present invention is further illustrated by the following examples:

Example 1 3 kg. of cowhides (moisture content: approximately 70%) fromwhich either the soluble protein has been removed by treating with a 5%aqueous solution of sodium chloride and then Washing with Water, or elsethe hair has been removed by liming, neutralizing with hydrochloric acidand then washing with water, are immersed in 3 l. of aqueous solutioncontaining trypsin (6 g.) and allowed to stand at the temperature of 25C. with occasional stirring for hours. Then the hydrogen ionconcentration of the enzyme solution is adjusted to pH 8 with causticsoda or boric acid buffer solution. The same result is obtained by usingpancreatin instead of trypsin. The amount of enzyme used is from about0.5 to 2.0%

of the substrate in accordance with its activity. During the treatmentwith anzyrne, the collagen does not dissolve at all and also noremarkable change is observed in its appearance. After removing thetrypsin by sufiicient rinsing in running water, the cowhide thus treatedwith enzyme is immersed in l. of water and the aqueous solution isadjusted to pH 2-3 at equilibrium by adding hydrochloric acid withstirring. For this purpose about 35 cc. of 12 N concentratedhydrochloric acid is required. By stirring at a temperature of 20-25 C.for about 24 hours, the cowhide is 100% dissolved to form a viscoussolution similar to gluten. If a mineral acid such as sulfuric acid,phosphoric acid etc. or an organic acid such as acetic acid, citric acidetc., is used instead of hydrohloric acid the same result is obtained.The viscous solution is filtered through a filter press using cloth andnon-fat cotton as a filter. The filtrate is neutralized by adding about56 cc. of 30% caustic soda solution thereto, the hydrogen ionconcentration is adjusted to pH 5-8, and the solution is allowed tostand for several hours, and a fibrous white precipitate is produced.This precipitate is washed thoroughly with water, collected byfiltration or centrifugation and dried in air. mately 700 g. of snowwhite collagen is obtained. Since the nitrogen content of thesupernatant liquid is nearly zero, it is obvious that the collagen fiberhas been completely reconstituted. And also, such collagen fiber can bereconstituted in 100% yield by dialysis of a 0.02 M disodium hydrogenphosphate solution or by adding an organic solvent such as acetone oralcohol in an amount sufficient to render the concentration of themixture about 30%, as well as by the above neutralization processsolubilized. The soluble collagen fiber thus obtained has the samephysico-chemical properties-viscosity, birefringent flow, specificrotation, denaturation temperature, shrinkage temperature, sedimentationconstant etc.-as the soluble collagen well known in the prior art. Thatis, my

solubilized collagen fiber has a molecular structure of rigid rod type,an intrinsic viscosity of 15, a uniform molecular length and diameter of3,000 A. and 13.6 A. respectively, a specific rotation of -415, asedimenta- Approxition constant of 30 (Svedberg unit), a denaturationtemperature of 37 C. and a shrinkage temperature of 60 C. When thereconstituted collagen fiber is observed under an electron microscope,it is seen that the collagen fiber has a cross striated pattern having aperiod of 700 A., which is also characteristic of the undissolved nativecollagen fiber.

Example 2.

3 kg. of cowhides (moisture content, approximately 70%) which have beenpretreated as described in Example 1 are added to 3 1. of aqueoussolution containing pepsin (6 g.) and its hydrogen ion concentration isadjusted to pH 2.0-2.5 with hydrochloric acid and is maintained at thetemperature of 25 C. with occasional stirring for 48 hours. Since theoptimum pH of pepsin is about 2, the enzyme treatment of the insolublecollagen and its dissolution in dilute acid are here accomplishedsimultaneously. However, the solubility of collagen in dilute acid isonly about 1%, so that the cowhides dissolve only partially. After thetreatment with enzyme for 48 hours, the volume of the solution isincreased to 100 l. by adding 0.005 N. hydrochloric acid and it isstirred constantly at the temperature of 25 C. for 24 hours, then allthe collagen is dissolved. The same result is obtained by using anotherinorganic acid or an organic acid as described in Example 1, instead ofhydrochloric acid. The method for reconstituting a fiber from thecollagen solution thus obtained is the same as that described inExample 1. That is, by neutralizing the solution with caustic soda andletting it stand for several hours, the collagen is precipitated. Afterwashing and drying the precipitate, about 700 g. of collagen fiber isobtained. The collagen fiber thus obtained by pepsin digestion isslightly different in its molecular properties from the soluble collagenobtained by the prior art and from the solubilized collagen obtained bytrypsin digestion as described in Example 1. That is, this collagenfiber is the same as the other two in its denaturation temperature,shrinkage temperature, specific rotation, sedimentation constant,homogeneity of molecular weight, etc., and, also, it has the samehelical structure of rigid rod type as the other two types; however, theintrinsic viscosity is 9.5, which is slightly less than in the other twoand, also, the molecular length is shorter than the other two byapproximately 200 A. in accordance with the measurement of birefringenceof flow. Thus, it is seen that this process cuts off a portion near theend of the natural collagen fiber molecule. When the soluble collagenthus obtained is suspended in water in the ratio of 1 part collagen to 2parts water by weight, and heated at a temperature of 60-70 C., thecollagen is denatured and dissolved in a few minutes. The collagensolution thus obtained is cooled to form a gel and then it is dried toproduce a gelatin. As compared with the gelatin produced by the priorart, the gelatin thus obtained is superior in the following respects:

1) As compared with the prior art liming method, only about one-fifth asmany hours are required for making gelatin.

(2) The yield is almost 100%.

(3) The physical properties such as jelly strength, setting point,melting point etc. are better than the best quality of product producedin the prior art method, and the best quality product constituted onlyabout 30% of the total product obtained in this prior art method.

(4) No concentration step is necessary, which cuts the heating cost inhalf; in the prior art method concentration after extraction isnecessary.

(5) The molecular weight of the gelatin is fairly uniform, being in theneighborhood of 120,000, while the prior art gelatin contains moleculesof widely distributed weights. The purity is much higher than in thegelatin of the prior art, since according to this invention the collagenfiber is dispersed in a molecular state as a solution and is thenreconstituted as a fiber; that is, the manufacturing process includesrecrystallization. Therefore, my gelatin is of especially high value forphotographic purpose.

When the collagen solution obtained as described in Examples 1 and 2 iscoated on a plastic plate and dried at room temperature, a clear film ofcollagen is obtained. This film is useful for medicine capsules, foodcasings, and the like.

When the collagen solution obtained as described in Example 1 isfiltered, extruded through a nozzle into a 2 M sodium chloride solutionat the temperature of 25 C. to be reconstituted into fibers, and wateris removed with acetone, and the resulting product tanned in a mixtureof 10% formalin and 0.02 M disodium hydrogen phosphate and dried in airor with acetone, a collagen yarn is obtained. Furthermore, if a solutionconsisting of the collagen solution to which has been added a gelatinobtained in the process described above is extruded through a nozzleinto a mixture of 0.02 M disodium hydrogen phosphate, 10% formalin and 2M sodium chloride to reconstitute fibers, a yarn having a moderateelasticity can be obtained. In reconstituted fibers made out of proteinsuch as albumin, casein or silk by previous processes the molecularstructure is changed in the course of manufacturing process; incontrast, the reconstituted fiber made out of collagen by this inventionis characterized by having the same molecular structure as the original.

The invention has been described in detail with particular reference topreferred embodiments thereof, but it will be understood that variationsand modifications can be efiected within the spirit and scope of theinvention as described hereinabove and as defined in the appendedclaims.

What is claimed is:

l. A process for making a solution of undenatured collagen from acollagen which is insoluble in dilute acid, alkali, and neutral saltsolutions, which comprises treating said insoluble collagen with ahydrolytic enzyme selected from the group consisting of trypsin andpepsin at a temperature below 60 C. and extracting the collagen with adilute acid of about pH 1 to about pH 4 at a temperature below 37 C.,whereby a collagen solution is produced.

2. The process of claim 1 in which the solution is then treated toreconstitute the collagen fiber.

3. The process of claim 2 in which the reconstituted fiber is suspendedin water, heated to a temperature of about 60 C. to 70 C. wherebydissolution takes place, then cooled to form a gel, and the gel is driedto a gelatin.

4. The process of claim 3 in which the hydrolytic enzyme is pepsin.

5. The process of claim 2 in which the collagen solution isreconstituted by extruding it through a nozzle into a 2 M sodiumchloride solution, water is removed with acetone, and the resultantfiber is tanned in a mixture of 10% formalin and 0.02 M disodiumhydrogen phosphate and then dried to produce a yarn.

6. The process of claim 5 in which the gelatin is dissolved in water andadmixed with a collagen solution prepared by treating a collageninsoluble in dilute acid, alkali and salt solutions with trypsin at atemperature below 60 C. and extracting the collagen with a dilute acidof about pH 1 to about pH 4 at a temperature below 37 C., and the mixedsolution is extruded into a mixed solution of 2 M sodium chloride, 0.02M disodium hydrogen phosphate and 10% formalin, and the resultant fiberis dried to form a yarn.

7. The process of claim 1 in which the solution is coated on a plasticplate and dried at room temperature to form a film.

8. A process for making a solution of undenatured col lagen from acollagen which is insoluble in dilute acid, alkali, and neutral saltsolutions which comprises treating said insoluble collagen with aproteolytic enzyme at a temperature below 60 C. and extracting thecollagen with a dilute acid of about pH 1 to about pH 4 at a temperaturebelow 37 0., whereby a collagen solution is produced.

9. A process for making a solution of undenatured collagen from acollagen normally insoluble in dilute acid, alkali and neutral saltsolutions, comprising the steps of treating said insoluble collagen withpancreatin at a temperature below the shrinkage temperature of saidcollagen; removing the trypsin; hydrolyzing the collagen; adjusting theaqueous solution to pH 1-4 with dilute acid and stirring at atemperature below the denaturation temperature of the collagen until acollagen solution is prodnced.

References Cited in the file of this patent UNITED STATES PATENTS2,377,854 Boyer et al. June 12, 1945 2,475,697 Cresswell July 12, 194910 2,598,608 Sale at al May 27, 1952 2,747,228 Braun May 29, 1956

1. A PROCESS FOR MAKING A SOLUTION OF UNDENATURED COLLAGEN FROM ACOLLAGEN WHICH IS INSOLUBLE IN DILUTE ACID, ALKALI, AND NEUTRAL SALTSOLUTIONS, WHICH COMPRISES TREATING SAID INSOLUBLE COLLAGEN WITH AHYDROLYTIC ENZYME SELECTED FROM THE GROUP CONSISTING OF TRYPSIN ANDPEPSIN AT A TEMPERATURE BELOW 60*C. AND EXTRACTING THE COLLAGEN WITH ADILUTE ACID OF ABOUT PH 1 TO ABOUT PH 4 AT A TEMPERATURE BELOW 37*C.,WHEREBY A COLLAGEN SOLUTION IS PRODUCED.